Relationships between DNA incorporation, mutant frequency, and loss of heterozygosity at the TK locus in human lymphoblastoid cells exposed to 3 '-azido-3 '-deoxythymidine

3'-Azido-3'-deoxythymidine (AZT), a thymidine analogue widely used in the t reatment of AIDS patients and for prevention of the onset of AIDS in HIV-se ropositive individuals, causes tumors in mice ex-posed as adults or in uter o. The purpose of this study was to investigate the potential mechanisms of AZT mutagenicity and carcinogenicity by quantifying the incorporation of A ZT into cellular DNA, measuring AZT-induced thymidine kinase (TK) mutant fr equencies (Mfs), and determining the percentage of loss of heterozygosity ( LOH) in spontaneous or AZT-induced TK mutants in the human lymphoblastoid c ell line, TK6. Cells were exposed to 300 mu M AZT for 0, 1, 3, or 6 days, o r to 0, 33, 100, 300, or 900 mu M AZT for 3 days (n = 5 flasks/group). The effects of exposure concentration on incorporation of AZT into cellular DNA were evaluated by an AZT radioimmunoassay, and the effects of duration and concentration of AZT exposure on the TK MfS were assessed by a cell-clonin g assay. AZT was incorporated into DNA in a dose-related manner at concentr ations up to 300 mu M, above which no further increase was observed. TK Mf increased with the extended duration and with incremental concentrations of AZT exposure. There was a positive correlation (P = 0.036, coefficient = 0 .903) between AZT-DNA incorporation and AZT-induced TK Mfs, suggesting that AZT incorporation into cellular DNA has a direct role in the genotoxicity of AZT. Southern blot analyses indicated that 84% (6.2 x 10(-6)/7.4 x 10(-6 )) of AZT-induced mutants were attributable to LOH, consistent with the kno wn mechanism of AZT as a DNA chain terminator. Considering the importance o f LOH in human carcinogenesis, AZT-induced LOH warrants further study.